Thermostabilization of proteins

ABSTRACT

Provided are compositions comprising a cocaine esterase (CocE) and a compound that thermostabilizes the CocE. Also provided are methods of thermostabilizing a cocaine esterase. Additionally provided are methods of treating a mammal undergoing a cocaine-induced condition. Methods of determining whether a compound is a thermostabilizing agent for a protein are also provided. Uses of the above-described compositions for the treatment of a cocaine-induced condition is additionally provided. Additionally provided is an isolated nucleic acid encoding a CocE polypeptide having the substitutions L169K and G173Q, and the CocE polypeptide encoded by that nucleic acid, and pharmaceutical compositions thereof. Further provided is the use of that composition for the manufacture of a medicament for the treatment of a cocaine-induced condition and for the treatment of a cocaine-induced condition.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of U.S. National Phaseapplication Ser. No. 12/667,895 filed 10 Jul. 2008 now U.S. Pat. No.8,637,009 issued 28 Jan. 2014; which claims the benefit of priority toInternational Application No. PCT/US08/69659 filed 10 Jul. 2008; whichclaims the benefit of U.S. Provisional Application Ser. No. 60/948,976filed 10 Jul. 2007 and U.S. Provisional Application Ser. No. 60/987,661filed 13 Nov. 2007; each of which is incorporated herein by reference inits entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant DA021416awarded by The National Institutes of Health, and Grant IIS-0324845awarded by the National Science Foundation. The Government has certainrights in the invention.

FIELD

The present application generally relates to anti-cocaine therapeutics.

BACKGROUND

Abuse of cocaine is an intractable social and medical problem that isresistant to remediation through pharmacotherapy. Cocaine acts to blockthe reuptake of monoamines, dopamine, norepinephrine, and serotonin thusprolonging and magnifying the effects of these neurotransmitters in thecentral nervous system (Benowitz, 1993). Cocaine toxicity is marked byboth convulsions and cardiac dysfunction (e.g., myocardial infarction,cardiac arrhythmias, increased blood pressure, stroke, or dissectinganeurysm, and increased myocardial oxygen demand), due to effects onneurotransmitter systems and myocardial sodium channel blockade (Baumanand DiDomenico, 2002; Wilson and Shelat, 2003; Knuepfer, 2003). Becauseof cocaine's ability to readily cross the blood brain barrier and itswidespread effects on the central and peripheral nervous systems,overdose can result in sudden death (see Bauman and DiDomenico, 2002 forreview).

Although the mechanism of cocaine's action is well understood, thisinformation has not yet resulted in the development of an effectiveantagonist of cocaine that could be used in abuse and overdosesituations. The rapid and pleiotropic effects of cocaine present acomplex problem for the treatment of acute cocaine toxicity (Carroll andKuhar, 1999). The two types of therapies that are available for thetreatment of opioid abuse, antagonism (e.g., naltrexone) and replacement(e.g., methadone), do not have parallels in the case of cocaine,although attempts at the latter are being considered (e.g., Grabowski etal., 2004). One approach is to prevent or reduce the cocaine fromreaching sites of action by administering either endogenous esterases,cocaine specific antibodies, or a catalytic antibody.

Naturally occurring cocaine is hydrolyzed at the benzoyl ester by serumbutyrylcholinesterase (BChE) to nontoxic ecgonine methyl ester andbenzoic acid. In the liver, carboxylesterase hCE-2 hydrolyzes the methylester to yield benzoylecgonine and methanol. The elimination half-lifeof cocaine in the blood ranges from 0.5 to 1.5 hr (Inaba, 1989). Therehave been a few attempts to use naturally occurring BChE or geneticallyengineered BChE to increase cocaine breakdown (see, e.g., Carmona etal., 2000; Xie et al., 1999; Sun et al., 2002a; Sun et al., 2002b;Duysen et al., 2002; Gao and Brimijoin S, 2004; Gao et al., 2005). Otherresearchers have utilized a monoclonal antibody, Mab 15A10, as acatalytic antibody to cocaine (see e.g., Landry et al, 1993; Mets etal., 1998), while others are exploring the use of cocaine vaccines (seee.g., Kosten et al., 2002).

A bacterium, Rhodococcus sp. MB 1, indigenous to the soil surroundingthe coca plant, has evolved the capacity to utilize cocaine as its solecarbon and nitrogen source. The bacterium expresses a cocaine esterase(CocE) that acts similarly to BChE to hydrolyze the benzoyl ester ofcocaine, yielding ecgonine methyl ester and benzoic acid (FIG. 1)(Bresler et al., 2000; Turner et al., 2002; Larsen et al., 2002). Thegene for CocE has been isolated and cloned (Bresler et al., 2000), andthe crystal structure of CocE has been determined (Turner et al., 2002;Larsen et al., 2002).

The purified enzyme (MW˜65 kDa) catalyzes cocaine very efficiently withMichaelis-Menten kinetics k_(cal)=7.2 s⁻¹ and K_(m)=640 nM (Turner etal., 2002; Larsen et al., 2002), nearly three orders of magnitudegreater than endogenous esterases and, most likely, would act quicklyenough to detoxify humans who have overdosed on cocaine (Landry et al.,1993; Mets et al., 1998). Additionally, the esterase also metabolizescocaethylene, a potent metabolite of cocaine and alcohol, almost asefficiently as it metabolizes cocaine (k_(cat)=9.4 s⁻¹ and K_(m)=1600nM) (Turner et al., 2002; Larsen et al., 2002).

One aspect of the Rhodococcus CocE that limits its usefulness is its lowthermostability—its t_(1/2) at 37° C. is about 15 minutes, whereas itst_(1/2) at 4° C. is >6 mo (PCT Patent Application PCT/US2007/015762,incorporated by reference herein). Thermostability was geneticallyengineered into CocE, with several mutant proteins having an increasedt_(1/2) at 37° C. up to ˜326 min (Id.).

There is a need for additional methods and compositions forthermostabilization of CocE. The present invention addresses that need.

SUMMARY

The inventors have discovered that certain compounds thermostabilizewild-type CocE, and further thermostabilize CocE mutants that werealready more thermostable than wild-type CocE.

Thus, the application is directed to compositions comprising a cocaineesterase (CocE) and a compound, where the CocE in the presence of thecompound is more thermostable than the CocE in the absence of thecompound.

The application is also directed to methods of thermostabilizing acocaine esterase (CocE). The methods comprise combining the CocE withthe compound

The application is additionally directed to methods of treating a mammalundergoing a cocaine-induced condition. The methods compriseadministering the above-described composition to the mammal in a mannersufficient to reduce the effects of the cocaine-induced condition on themammal.

Also, the application is directed to methods of treating a mammalundergoing a cocaine overdose. The methods comprise administering theabove-described composition to the mammal in a manner sufficient toreduce the effects of the cocaine on the mammal.

The application is further directed to methods of treating a mammalhaving a cocaine dependence. The methods comprise administering theabove-described composition to the mammal in a manner sufficient toreduce the effects of the cocaine dependence on the mammal.

Additionally, the application is directed to methods of determiningwhether a compound is a thermostabilizing agent for a protein. Themethods comprise measuring the thermostability of the protein with andwithout the compound. With these methods, a compound that causes theprotein to be more thermostable is a thermostabilizing agent for theprotein.

The application is also directed to the use of the above compositionsfor the manufacture of a medicament for the treatment of acocaine-induced condition.

The application is additionally directed to the use of the abovecompositions for the treatment of a cocaine-induced condition.

It has also been discovered that the CocE mutant L169K/G173Q has anunexpectedly high degree of thermostablity. See Example 5.

Thus, the application is additionally directed to an isolated nucleicacid encoding a CocE polypeptide comprising an amino acid sequence thathas at least 85% sequence identity with the polypeptide of SEQ ID NO:1,wherein the encoded CocE polypeptide has (a) the substitutions L169K andG173Q, and (b) esterase activity with increased thermostability at 37°C. as compared to wild-type CocE.

The application is also directed to the CocE polypeptide comprising anamino acid sequence that has at least 85% sequence identity with thepolypeptide of SEQ ID NO:1, wherein the encoded CocE polypeptide has thesubstitutions L169K and G173Q, and esterase activity with increasedthermostability at 37° C. as compared to wild-type CocE. Compositionscomprising the polypeptide in a pharmaceutically acceptable carrier arealso provided.

In additional embodiments, the application is directed to a method oftreating a mammal undergoing a cocaine-induced condition. The methodcomprises administering the composition described immediately above tothe mammal in a manner sufficient to reduce the effects of thecocaine-induced condition on the mammal. The use of that composition forthe manufacture of a medicament for the treatment of a cocaine-inducedcondition is also provided, as is the use of that composition for thetreatment of a cocaine-induced condition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the metabolism of cocaine catalyzed by Rhodococcus cocaineesterase (CocE).

FIG. 2A-C are graphs of cocaine denaturation over time in the presenceand absence of CocE.

FIG. 2A is a graph of cocaine denaturation over time in the absence ofCocE.

FIG. 2B is a graph of cocaine denaturation over time in the presence of62.5 ng/ml CocE.

FIG. 2C is a graph of cocaine denaturation over time in the presence of250 ng/ml CocE.

FIG. 3A shows the metabolism of 4-nitrophenyl acetate (4NPA) catalyzedby CocE.

FIG. 3 B shows a graph of 4NPA denaturation over time in the absence ofCocE.

FIG. 3C shows a graph of 4NPA denaturation over time in the presence of62.5 ng/ml CocE.

FIG. 3D shows a graph of 4NPA denaturation over time in the presence of250 ng/ml CocE.

FIG. 4 is a photograph of a nondenaturing gel showing the aggregation ofCocE after 1 h incubation under various conditions and in the presenceor absence of substrates or products.

FIG. 5 is a graph of a spectrophotometric analysis of cocainestabilization during cleavage of 4NPA in the presence of variousconcentrations of cocaine.

FIG. 6A is a graph showing a kinetic spectrophotometric analysis ofbenzoic acid stabilization of CocE.

FIG. 6B is a graph showing a kinetic spectrophotometric analysis ofecgonine methyl ester stabilization of CocE.

FIG. 6C is a graph showing a kinetic spectrophotometric analysis ofsodium acetate stabilization of CocE.

FIG. 6D is a graph showing a kinetic spectrophotometric analysis ofbenzoic acid stabilization of CocE at higher concentrations of benzoicacid.

FIG. 7A-E are graphs, chemical structures, a diagram of an enzymaticreaction and a photograph of a nondenaturing gel showing thethermostabilization of CocE by phenylboronic acid (PBA).

FIG. 7A is a chemical structure of phenylboronic acid (PBA).

FIG. 7B is a graph showing the thermostabilization of CocE byphenylboronic acid (PBA).

FIG. 7C is a reaction scheme showing the enzymatic cleavage of4-nitrophenylacetate.

FIG. 7D is a photograph of a nondenaturing gel. FIG. 7E is adensitometry curve.

FIG. 8 is a graph showing benzoic acid thermostabilization of CocE.

FIG. 9 is a graph showing the results of a screening of 40 compounds forthe ability to thermostabilize CocE, along with the chemical structuresof the most effective compounds.

FIG. 10A-C show the results of studies on the ability of a compound toinhibit CocE.

FIG. 10A is the structure of compound 6031818.

FIG. 10B is a graph showing the compound is a weak inhibitor of4-nitrophenyl acetate cleavage.

FIG. 10C is a graph showing the compound does not inhibit cocainecleavage at all.

FIG. 11A-C are graphs and the structure of compound 6031818 showing theability of that compound to thermostabilize CocE when 4NPA or cocaineare used as substrates.

FIG. 11A is the structure of compound 6031818.

FIG. 11B shows the ability of that compound to thermostabilize CocE when4NPA is used as a substrate.

FIG. 11C shows the ability of that compound to thermostabilize CocE whencocaine is used as a substrate.

FIG. 12A-C are graphs and the structure of compound 6169221 showingresults of studies on the ability of that compound to inhibit CocE.

FIG. 12A shows the structure of compound 6169221.

FIG. 12B shows the compound did not inhibit 4-nitrophenyl acetatecleavage.

FIG. 12C shows the compound did not inhibit cocaine cleavage.

FIG. 13A-C are graphs and the structure of compound 6169221 showing theability of that compound to thermostabilize CocE when 4NPA or cocaineare used as substrates.

FIG. 13A is the structure of compound 6169221.

FIG. 13B shows the ability of that compound to thermostabilize CocE when4NPA is used as a substrate.

FIG. 13C shows the ability of that compound to thermostabilize CocE whencocaine is used as a substrate.

FIG. 14A-C are graphs and the structure of compound 5804236 showingresults of studies on the ability of that compound to inhibit CocE.

FIG. 14A is the chemical structure of compound 5804236.

FIG. 14B is a graph showing the compound did not inhibit 4-nitrophenylacetate cleavage.

FIG. 14C is a graph showing the compound did not inhibit cocainecleavage.

FIG. 15A-C are graphs and the structure of compound 5804236 showing theability of that compound to thermostabilize CocE when 4NPA or cocaineare used as substrates.

FIG. 15A is the chemical structure of compound 5804236.

FIG. 15B is a graph showing the ability of that compound tothermostabilize CocE when 4NPA is used as a substrate.

FIG. 15C is a graph showing the ability of that compound tothermostabilize CocE when cocaine is used as a substrate.

FIG. 16A-D are graphs that further characterize, by circular dichroism,the stability of wild type CocE and mutant T172R.

FIG. 16A is a circular dichroism spectrum of CocE WT.

FIG. 16B is a graph showing the melting temperature of CocE WT.

FIG. 16C is a circular dichroism spectrum of T172R mutant.

FIG. 16D is a graph showing the melting temperature of T172R mutant.

FIG. 17A-D are graphs that further characterize, by circular dichroism,the stability of wild type CocE in the presence of benzoic acid orphenylboronic acid.

FIG. 17A is a circular dichroism spectrum of CocE WT in the presence ofbenzoic acid.

FIG. 17B is a graph showing the melting temperature of CocE WT in thepresence of benzoic acid.

FIG. 17C is a circular dichroism spectrum of CocE WT in the presence ofphenylboronic acid.

FIG. 17D is a graph showing the melting temperature of CocE WT in thepresence of phenylboronic acid.

FIG. 18A-D are graphs that further characterize, by circular dichroism,the stability of CocE mutant L169K in the absence or presence ofphenylboronic acid.

FIG. 18A is a circular dichroism spectrum of the L169K mutant.

FIG. 18B is a graph showing the melting temperature of the L169K mutant.

FIG. 18C is a circular dichroism spectrum of the L169K mutant in thepresence of phenylboronic acid.

FIG. 18D is a graph showing the melting temperature of the L169K mutantin the presence of phenylboronic acid.

FIG. 19A-D are graphs that further characterize, by circular dichroism,the stability of wild type CocE in the presence or absence of compound6031818.

FIG. 19A is circular dichroism spectra of PBS, compound 6031818, andCocE in the presence of compound 6031818.

FIG. 19B is a graph showing the melting temperature of CocE WT in thepresence of 6031818.

FIG. 19C is circular dichroism spectra of CocE WT in the presence of6031818.

FIG. 19D shows the irreversible melting of CocE WT in the presence of6031818.

FIG. 20A is a photograph of a non-denaturing gel showing the results ofan analysis of wild-type CocE in the presence of various small moleculesafter incubation at 37° C. for various time points.

FIG. 20B is a photograph of a non-denaturing gel showing the results ofan analysis of CocE mutant L169K in the presence of various smallmolecules after incubation at 37° C. for various time points.

FIG. 21A is a graph showing the results of a spot densitometry half-lifeanalysis of the gels shown in FIG. 20.

FIG. 21B is a table showing the results of a spot densitometry half-lifeanalysis of the gels shown in FIG. 20.

FIG. 22A-B are diagrams showing the structure of cocaine esterase.

FIG. 22A shows cocaine esterase is composed of three distinct domains,as demarcated.

FIG. 22B shows point mutations predicted by computational methodssuperimposed on the crystal structure of wt-CocE. Coordinates wereobtained from the RCSB database (pdb:1JU4) by Larsen et al (2002).Structure models were generated and rendered with PyMol (DeLanoScientific, Palo Alto, Calif.).

FIG. 23 is a graph showing the decay in CocE activity at 37° C. 50 ng/mlwild-type CocE and the mutants were incubated at 37° C. and activitymeasured (Xie et al., 1999) over time. Half-lives were measured fromresulting curves. Wild-type CocE, T172R, L169K and T172R/G173Q showed 12min, 46 min, 274 min and 326 min half-lives, respectively.

FIG. 24 is a graph showing that DTT inhibits wt-CocE in aconcentration-dependent manner with an IC₅₀˜390 μM.

FIG. 25 is a graph showing that the presence of DTT (10 mM) shortens thein vitro τ_(inact) in wt-CocE.

FIG. 26 is a graph showing temperature-dependent decay in esteraseactivity. 50 ng/ml wild-type CocE and the mutants were pre-incubated for30 min at temperatures indicated (° C.) and activity measured (Xie etal., 1999). The activity of each mutant remaining (as a percentage ofthe maximal activity, V_(max), without preincubation) followingpre-incubation are illustrated. Wild-type CocE (open bars) appears toinactivate between 30-35° C. whereas T172R/G173Q (shaded bars) and L169K(solid) both display enhance thermal stability (inactivation at 40-45°C.).

FIG. 27 is a graph showing protective effects of CocE againstcocaine-induced toxicity. CocE (1 mg) was administered intravenously 1min before cocaine administration (mg/kg, i.p.). Dose-response curves ofcocaine-induced lethality in the absence or presence of CocE or mutantswere plotted. Each data point represents the percentage of mice (n=6 foreach dosing condition) exhibiting cocaine-induced lethality.

FIG. 28A-C are graphs showing the time course of protective effects ofCocE against cocaine toxicity. Each data point represents the percentageof mice (n=6 for each dosing condition) exhibiting cocaine-inducedlethality.

FIG. 28A is a graph showing 0.1 mg i.v. CocE or mutants administered atdifferent time points before cocaine administration (180 mg/kg, i.p.).

FIG. 28B is a graph showing 0.3 mg i.v. CocE or mutants administered atdifferent time points before cocaine administration (180 mg/kg, i.p.).

FIG. 28C is a graph showing 1 mg i.v. CocE or mutants administered atdifferent time points before cocaine administration (180 mg/kg, i.p.).

FIG. 29 is a graph showing the estimated duration of protection for 50%lethality. Time required to reach 50% lethality for CocE and each mutantwas measured from the data in FIG. 28 and was plotted against dosage.

FIG. 30 is a diagram showing an overview of CocE and thermostabilizingmutations. The H2 and H3 helices of CocE are shown as coils, and theremainder of CocE as a molecular surface. The molecule of DTT observedbound in our crystal structures indicates the relative position of theactive site, in a cavity adjacent to the H2 helix. The three identifiedstabilizing mutations are indicated. The T172R mutation leads to van derWaals interactions between R172 (H2 helix) and F189 (H3 helix). TheG173Q mutation bridges the active site cleft with a new hydrogen bond(dashed lines). The L169K mutation impinges on the active site. Both DTTand glycerol are observed in the active site in this crystal structure,with K169 exhibiting multiple conformations and forming hydrogen bondswith glycerol. Note that L169 is poorly ordered in the native structure.K169 forms additional direct contacts with Y44. Glycerol binds where thetropane ring of cocaine is expected to bind, while DTT occupies thebenzyl moiety binding site. Structure model was generated and renderedwith PyMol (DeLano Scientific, Palo Alto, Calif.).

FIG. 31A-F are diagrams showing the structure of the thermal stablemutants. The high resolution crystal structures of T172R (B), G173Q (D)and L169K (F) are compared to the structures of wt-CocE (A, C, and E,respectively). The overall effect of the mutants appears to result fromenhanced interactions between the helix 1 and 2 of domain II (R172 andF189) or interdomain interactions (Q173 with P44 and K169 with theactive site). The structures of L169K in comparison with wt-CocE in thepresence of 2-oxo-dioxolane butyryl carbonate (DBC) in the active siteillustrates and enhanced interaction of the lysine residue with a waterand the active site. The 2F_(o)F_(c) electron densities were contouredat 1 sigma.

FIG. 31A high resolution crystal structures of wt-CocE.

FIG. 31B high resolution crystal structure of T172R.

FIG. 31C high resolution crystal structures of wt-CocE.

FIG. 31D high resolution crystal structure of G173Q.

FIG. 31E high resolution crystal structures of wt-CocE.

FIG. 31F high resolution crystal structure of L169K.

FIG. 32 is a diagram showing the stabilization of the H1-H2 loop inDomain II by R172. The substitution of arginine for threonine at residue172 stabilizes helix 1 and helix 2 through enhancing interactions withF189. Although both the ‘in” (dark) and “out” (light) conformations canbe found in the wt-CocE structures, only the “out” conformation is foundwith T172R.

FIG. 33A-B are diagrams showing a comparison of DBC and phenyl boronicacid in the active site of wt-CocE. DBC was found covalently bound tothe active site serine 117 of crystals grown from protein isolated withDTT.

FIG. 33A is an omit map of the DBC molecule showing carbon density at 5sigma, oxygen density at 12 sigma, and sulfur density at 20 sigma.

FIG. 33B depicts the previously reported binding site of thetransition-state analog phenyl boronic acid (pdb:1JU3), which isanalogous to the DBC binding site. Omit map density for the L169K mutantis shown, which coordinates a water molecule which hydrogen bonds to theDBC ring.

FIG. 34A is a graph showing catalytic parameters of wt-CocE and twodouble mutants.

FIG. 34B is a table showing catalytic parameters of wt-CocE and twodouble mutants.

FIG. 35A is a graph showing in vitro loss of activity of wt-CocE, twoCocE mutants (T172R and L169K), and a double mutant combining T172R andG173Q mutations.

FIG. 35B is a graph showing in vitro loss of activity of a double mutantcombining two single mutations, L169K and G173Q.

FIG. 36 is a graph showing protection of mice from a lethal cocaine doseby the CocE L169K/G173Q mutant.

FIG. 37A-B are graphs showing a time course of protection of mice from alethal cocaine dose by the CocE L169K/G173Q mutant.

FIG. 37A is a graph showing a time course of protection of mice from alethal cocaine dose by 1 mg CocE L169K/G173Q mutant; 1 mg CocE L169Kmutant; 1 mg CocE T172R/G173Q mutant; and 1 mg wild type CocE.

FIG. 37B is a graph showing a time course of protection of mice from alethal cocaine dose by 1 mg CocE L169K/G173Q; 0.32 mg CocE L169K/G173Q;and 0.1 mg CocE L169K/G173Q.

DETAILED DESCRIPTION

The inventors have discovered that certain compounds thermostabilizewild-type CocE, and further thermostabilize CocE mutants that werealready more thermostable than wild-type CocE. See Examples 1-3.

Thus, the application is directed to compositions comprising a cocaineesterase (CocE) and a compound, where the CocE in the presence of thecompound is more thermostable than the CocE in the absence of thecompound.

The resulting increase in thermostability of the CocE in the presence ofthe compound increases the half-life of the enzyme at 37° C. at leastabout 5 minutes, preferably at least about 10, 15, 20, 25, 30, 35 or 40minutes, or more.

Thermostability of a given polypeptide can be assessed by a variety ofmethods known to the art, including for example measuring circulardichroism (CD) spectroscopy (as in, e.g., Example 2, below) ordifferential scanning calorimeter. See also PCT Patent ApplicationPCT/US2007/015762, published as WO/2008/008358, incorporated byreference. Preferably, thermostability is determined by measuring enzymeactivity over time at a low and high temperature with and without thecompound, to determine whether, and to what degree, the compound causesthe enzyme to maintain enzymatic activity at the higher temperature morethan without the compound. A preferred low temperature is roomtemperature (i.e., ˜25° C.); a preferred high temperature is 37° C.However, any temperature ranges can be used. The skilled artisan coulddetermine the best temperature range for any particular applicationwithout undue experimentation.

As used herein, a CocE is an enzyme having an amino acid sequence atleast 80% identical to SEQ ID NO:1 and is capable of specificallycatalyzing the cleavage of cocaine into ecgonine methyl ester andbenzoic acid. Preferably, the CocE has an amino acid sequence at least90%, more preferably, 95%, even more preferably 99% identical to SEQ IDNO:1. In some preferred embodiments, the CocE has an amino acid sequenceidentical to SEQ ID NO:1.

In other embodiments, the CocE has a mutation, such as those describedin PCT Patent Application PCT/US2007/015762, including mutants that haveincreased thermostability over the wild type (SEQ ID NO:1) and mutantsthat do not. Preferred mutants are those where the CocE has the aminoacid sequence of SEQ ID NO:1 except for the substitution L163V, V225I,I218L, A310D, A149S, S159A, S265A, S56G, W220A, S140A, F189L, A193D,T254R, N42V, V262L, L508G, Y152H, V160A, T172R, Y532F, T74S, W285T,L146P, D533S, A194R, G173Q, C477T, K531A, R41I, L119A, K46A, F84Y,T172R/G173Q, L169K, F189A, N197K, R182K, F189K, V190K, Q191K, or A194K,or any combination of these mutated amino acid residues. In some ofthese embodiments, the CocE has the amino acid sequence of SEQ ID NO:1except for the substitution T172R, S159A, N197K, L169K, F189K, G173Q, orT172R/G173Q. In other of these embodiments, the CocE has the amino acidsequence of SEQ ID NO:1 except for the substitution L169K/G173Q.

A compound within the scope of these embodiments can increase thethermostability of the wild type and/or a mutant CocE described above.

The CocE can also be pegylated or otherwise treated to increase theduration of action, heat stability, and/or decrease immunogenicity.Pegylation can further enhance the thermostability of the CocE-compoundcompositions and, when used in vivo, increase serum half life bydecreasing renal clearance, proteolysis, macrophage uptake, andimmunological response.

The CocE-compound compositions can be encapsulated into red blood cells(RBC) so as to increase the duration of action and heat stability, anddecrease immunogenicity. In preferred compositions, the compound is:

More preferably, the compound is

Even more preferably, the compound is

In other preferred embodiments, the compound is

The compound can also be

The compound can additionally be

Additionally, the compound can be

Further, the compound can be

The compound can also be

The compositions of the present invention can comprise more than one ofany of the above-identified compounds that thermostabilize CocE.

In some embodiments of these compositions, in particular where they areused for therapeutic purposes, the composition is in a pharmaceuticallyacceptable carrier.

The CocE-compound compositions described herein can be formulated by anyconventional manner using one or more pharmaceutically acceptablecarriers and/or excipients (see e.g., Gennaro (2005) Remington theScience and Practice of Pharmacy 21st ed. Lippincott Williams & Wilkins,ISBN 0781746736). Such formulations will contain a therapeuticallyeffective amount of the CocE-compound compositions, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the subject. Theformulation should suit the mode of administration. The CocE-compoundcompositions of use with the current application can be formulated byknown methods for administration to a subject using several routes whichinclude, but are not limited to, parenteral, pulmonary, oral, topical,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, epidural, ophthalmic, buccal, and rectal. The CocE-compoundcompositions can also be administered in combination with one or moreadditional agents disclosed herein and/or together with otherbiologically active or biologically inert agents. Such biologicallyactive or inert agents can be in fluid or mechanical communication withthe agent(s) or attached to the agent(s) by ionic, covalent, Van derWaals, hydrophobic, hydrophilic or other physical forces.

The CocE-compound compositions described herein can be administeredparenterally, including by intravenous, intramuscular, subcutaneous, orintraperitoneal injections. Excipients, commonly used in the parenteraldelivery of small drug molecules, including solubility enhancers,osmotic agents, buffers, and preservatives, can also be included inbiomolecule formulations. Inclusion of antiaggregation andantiadsorption agents, such as surfactants and albumin, when formulatingand delivering biomolecules can add increased stability and decrease therisk of the active biomolecule interacting with an interface, which canlead to unfolding, aggregation, and/or precipitation. The CocE-compoundcompositions can be lyophilized for added stability during storage, andre-processed before parenteral administration.

Pulmonary delivery of the CocE-compound compositions is alsocontemplated. Additionally, controlled-release (or sustained-release)preparations can be formulated to extend the activity of the mutant CocEpolypeptide and reduce dosage frequency, as is known in the art.

The CocE-compound compositions can be encapsulated and administered in avariety of carrier delivery systems. Examples of carrier deliverysystems for use with mutant CocE polypeptides described herein includemicrospheres (see e.g., Varde & Pack (2004) Expert Opin. Biol. 4(1)35-51), hydrogels (see generally, Sakiyama et al. (2001) FASEB J. 15,1300-1302), polymeric implants (see generally, Teng et al. (2002) Proc.Natl. Acad. Sci. U.S.A. 99, 3024-3029), smart polymeric carriers (seegenerally, Stayton et al. (2005) Orthod Craniofacial Res 8, 219-225; Wuet al. (2005) Nature Biotech (2005) 23(9), 1137-1146), and liposomes(see e.g., Galovic et al. (2002) Eur. J. Pharm. Sci. 15, 441-448; Wagneret al. (2002) J. Liposome Res. 12, 259-270).

The application is also directed to methods of thermostabilizing acocaine esterase (CocE). The methods comprise combining the CocE withthe compound

Preferably, the compound is

More preferably, the compound is

In other preferred embodiments, the compound is

The compound can also be

The compound can additionally be

Additionally, the compound can be

Further, the compound can be

The compound can also be

In these methods, the CocE can be combined with more than onethermostabilizing compound, for example one of the compounds describedabove, or with any other compound.

Preferably, the CocE comprises an amino acid sequence at least 90%identical to SEQ ID NO:1; more preferably at least 95% identical to SEQID NO:1; even more preferably at least 99% identical to SEQ ID NO:1. Inother preferred embodiments, the CocE comprises the amino acid sequenceof SEQ ID NO:1. In additional embodiments, the CocE is a thermostablemutant of a wild-type CocE having the amino acid sequence of SEQ IDNO:1. Preferred examples of such thermostable mutants is the CocE havingthe amino acid sequence of SEQ ID NO:1 except for the substitutionT172R, S159A, N197K, L169K, F189K, G173Q, or T172R/G173Q. Additionally,the CocE can have the amino acid sequence of SEQ ID NO:1 except for thesubstitution L169K/G173Q.

The methods of these embodiments can be performed where the CocE is invitro, for example to thermostabilize CocE upon purification or duringstorage. Preferably, however, the CocE is in a living mammal. Towardthose embodiments, an aspect of this application is directed towardcatalytic degradation approaches to anti-cocaine therapeutics. Providedare treatments, both prophylactic and therapeutic, of cocaine-inducedconditions through the administration of thermostable, esterase-active,CocE-compound compositions to a subject in need thereof. It is theincrease in thermostability provided by the thermostabilizing compoundthat enables a much more rapid and effective response to symptoms ofcocaine toxicity that sets the CocE-compound compositions describedabove apart from other treatment options.

The application is additionally directed to methods of treating a mammalundergoing a cocaine-induced condition. The methods compriseadministering the above-described CocE-compound composition to themammal in a manner sufficient to reduce the effects of thecocaine-induced condition on the mammal.

A determination of the need for treatment will typically be assessed bya history and physical exam consistent with the cocaine-inducedcondition. It is contemplated that the present methods can be used fortreatment of any cocaine-induced condition including, but are notlimited to, cocaine overdose, cocaine toxicity, and cocaine dependenceand/or addiction. The diagnosis of such conditions is within the skillof the art. For example, the diagnosis of cocaine toxicity can includeassessment of convulsions, grand-mal seizures, cardiac arrest,myocardial infarction, cardiac arrhythmias, increased blood pressure,stroke, drug-induced psychosis, dissecting aneurysm, and increasedmyocardial oxygen demand. As another example, in the case of cocainedependence and/or addiction, withdrawal symptoms include subjectivesensations of mild to severe dysphora, depression, anxiety, orirritability. Subjects with an identified need of therapy include thosewith a diagnosed cocaine-induced condition, an indication of acocaine-induced condition, and subjects who have been treated, are beingtreated, or will be treated for a cocaine-induced condition. Thesemethods can be used to treat any mammal, including, but not limited to,rodents, rabbits, guinea pigs, horses, cows, dogs, cats, sheep and pigs,and most preferably humans.

An effective amount of the CocE-compound compositions described hereinis generally that which can reduce the cocaine-toxicity or the severityof a cocaine-induced condition. Reduction in severity includes, forexample, an arrest or a decrease in symptoms, physiological indicators,biochemical markers, or metabolic indicators. When used in the methodsof the invention, a therapeutically effective amount of CocE-compoundcompositions described herein can be employed in pure form or, wheresuch forms exist, in pharmaceutically acceptable salt form and with orwithout a pharmaceutically acceptable excipient. For example,CocE-compound compositions can be administered at a reasonablebenefit/risk ratio applicable to any medical treatment, in an amountsufficient to substantially reduce the cocaine concentration in theblood and/or tissues of the subject.

Toxicity and therapeutic efficacy of CocE-compound compositions can bedetermined by standard pharmaceutical procedures in cell cultures and/orexperimental animals for determining the LD50 (the dose lethal to 50% ofthe population) the ED50, (the dose therapeutically effective in 50% ofthe population), or other parameters.

The amount of CocE-compound compositions that can be combined with apharmaceutically acceptable carrier to produce a single dosage form willvary depending upon the host treated and the particular mode ofadministration. It will be appreciated by those skilled in the art thatthe unit content of agent contained in an individual dose of each dosageform need not in itself constitute a therapeutically effective amount,as the necessary therapeutically effective amount could be reached byadministration of a number of individual doses. Administration of theCocE-compound composition can occur as a single event or over a timecourse of treatment. For example, a CocE-compound composition can beadministered daily, weekly, bi-weekly, or monthly. For some conditions,treatment could extend from several weeks to several months or even ayear or more.

The specific therapeutically effective dose level for any particularsubject will depend upon a variety of factors including thecocaine-induced condition being treated and the severity of thecocaine-induced condition; activity of the mutant CocE polypeptideemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration;the route of administration; the plasma half-life of the mutant CocEpolypeptide; the rate of excretion of the mutant CocE polypeptideemployed; the duration of the treatment; drugs used in combination orcoincidental with the mutant CocE polypeptide employed; and like factorswell known in the medical arts (see e.g., Koda-Kimble et al. (2004)Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams &Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics,4th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Shama (2004)Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill/Appleton &Lange, ISBN 0071375503). It will be understood by a skilled practitionerthat the total daily usage of the CocE-compound compositions for use inembodiments of the invention disclosed herein can be decided withoutundue experimentation by the attending physician within the scope ofsound medical judgment.

The CocE-compound compositions described herein can also be used incombination with other therapeutic modalities. Thus, in addition to thetherapies described herein, one can also provide to the subject othertherapies known to be efficacious for particular cocaine-inducedconditions.

Thus, in some embodiments of these methods, the mammal is addicted tococaine. In other embodiments, the mammal is undergoing a cocaineoverdose.

The CocE for these methods can be a thermostable mutant of a wild-typeCocE having the amino acid sequence of SEQ ID NO:1. Preferred suchmutants have the amino acid sequence of SEQ ID NO:1 except for thesubstitution T172R, S159A, N197K, L169K, F189K, G173Q, T172R/G173Q, orL169K/G173Q. CocE mutants having more than one mutation, preferably morethan one thermostabilizing mutation, are also useful for these treatmentmethods.

These methods are not limited to the use of any particularthermostabilizing compound. Preferably, the compound is

More preferably, the compound is

In other preferred embodiments, the compound is

The compound can also be

The compound can additionally be

Additionally, the compound can be

Further, the compound can be

The compound can also be

In these methods, the CocE can be combined with more than onethermostabilizing compound, for example one of the compounds describedabove, or with any other compound.

The application is also directed to methods of treating a mammalundergoing a cocaine overdose. The methods comprise administering theabove-described composition to the mammal in a manner sufficient toreduce the effects of the cocaine on the mammal.

These methods can be used to treat any mammal, including, but notlimited to, rodents, rabbits, guinea pigs, horses, cows, dogs, cats,sheep and pigs, and most preferably humans.

As with the treatment methods described above, the composition can beadministered by any method known in the art. The skilled artisan coulddetermine the best mode of administration for any particular individualwithout undue experimentation. In some preferred embodiments, theCocE-compound composition is administered to the mammal intravenously.

The CocE for these methods can be a thermostable mutant of a wild-typeCocE having the amino acid sequence of SEQ ID NO:1. Preferred suchmutants have the amino acid sequence of SEQ ID NO:1 except for thesubstitution T172R, S159A, N197K, L169K, F189K, G173Q, or T172R/G173Q.In another thermostable mutant, the CocE has the amino acid sequence ofSEQ ID NO:1 except for the substitution L169K/G173Q. CocE mutants havingmore than one mutation, preferably more than one thermostabilizingmutation, are also useful for these treatment methods.

These methods are not limited to the use of any particularthermostabilizing compound. Preferably, the compound is

More preferably, the compound is

In other preferred embodiments, the compound is

The compound can also be

The compound can additionally be

Additionally, the compound can be

Further, the compound can be

The compound can also be

In these methods, the CocE can be combined with more than onethermostabilizing compound, for example one of the compounds describedabove, or with any other compound.

The invention is further directed to methods of treating a mammal havinga cocaine dependence. The methods comprise administering theabove-described composition to the mammal in a manner sufficient toreduce the effects of the cocaine dependence on the mammal.

These methods can be used to treat any mammal, including, but notlimited to, rodents, rabbits, guinea pigs, horses, cows, dogs, cats,sheep and pigs, and most preferably humans.

As with the treatment methods described above, the composition can beadministered by any method known in the art. The skilled artisan coulddetermine the best mode of administration for any particular individualwithout undue experimentation. In some preferred embodiments, theCocE-compound composition is administered to the mammal intravenously.

The CocE for these methods can be a thermostable mutant of a wild-typeCocE having the amino acid sequence of SEQ ID NO:1. Preferred suchmutants have the amino acid sequence of SEQ ID NO:1 except for thesubstitution T172R, S159A, N197K, L169K, F189K, G173Q, or T172R/G173Q.In another thermostable mutant, the CocE has the amino acid sequence ofSEQ ID NO:1 except for the substitution L169K/G173Q. CocE mutants havingmore than one mutation, preferably more than one thermostabilizingmutation, are also useful for these treatment methods.

These methods are not limited to the use of any particularthermostabilizing compound. Preferably, the compound is

More preferably, the compound is

In other preferred embodiments, the compound is

The compound can also be

The compound can additionally be

Additionally, the compound can be

Further, the compound can be

The compound can also be

In these methods, the CocE can be combined with more than onethermostabilizing compound, for example one of the compounds describedabove, or with any other compound.

Additionally, the application is directed to methods of determiningwhether a compound is a thermostabilizing agent for a protein. Themethods comprise measuring the thermostability of the protein with andwithout the compound. With these methods, a compound that causes theprotein to be more thermostable is a thermostabilizing agent for theprotein. Preferably, the protein is a CocE.

With these methods, the protein can be isolated (i.e., in a test tubeoutside of a living cell), or produced by cells in culture or in vivofor the thermostability determination.

The activity of the isolated protein with and without the compound canbe measured at one or more temperatures to determine the thermostabilityof the protein imparted by the compound. The temperature at which theactivity assay is performed determines the degree of thermostabilitydetection. Thus, initial screening can, for example, be performed at 30°C., and after initial compound selection, screening can be performedwith incrementally increasing temperatures (for example, 34° C., 37° C.,40° C., 42.5° C., 45° C., etc.), until a compound of suitablethermostability is achieved. The incremental temperature increases aredetermined empirically during the procedure, and are affected by thenumber of hits at particular temperatures and the determined Tm of theinitial compounds.

Where the protein is CocE, detection of esterase activity can beperformed using a variety of methods, where substrates generally arecoupled to a specific detection system. Appropriate substrates for usein determining esterase activity can include cocaine, tritiated (³H)cocaine, cocaine substrate derivatives such as a thio-cocainederivative, and/or substrates that report general esterase activity suchas 4-nitrophenyl acetate. The detection system can be directly coupledto the specifics of the substrate, for example: cleavage of unmodifiedcocaine can be detected by monitoring changes in cocaine absorbance at240 nm, or by monitoring pH changes that result from the accumulation ofthe acidic benzoic acid product, or through the use of cocaine aptamers(see e.g., Stojanovic, M. N., de Prada, P. & Landry, D. W. (2001) J AmChem Soc 123, 4928-4931; Stojanovic, M. N. & Landry, D. W. (2002) J AmChem Soc 124, 9678-9679) by monitoring changes in fluorescence upondegradation of cocaine; cleavage of tritiated (³H) cocaine can bedetected by acidification and detection of tritiated benzoic acidproduct through separation by chromatography; cleavage of cocainederivatives such as thio-cocaine can be monitored by the detection ofreactive sulfhydryl groups, through the addition of Ellman's reagent anddetermination of absorbance changes at 412 nm, or by the addition andvisualization of precipitating sulfhydryl reacting heavy metals;cleavage of 4-nitrophenyl acetate can be detected by monitoring changesin absorbance at 420 nm (see e.g., Halgasova, N. et al. (1994) Biochem J298 Pt 3, 751-755; O'Conner, C. J. & Manuel, R. D. (1993) J Dairy Sci.76, 3674-3682). See also PCT Publication WO/2008/008358 for furtherelaboration of the above.

Protein-compound compositions identified through the above procedures,or a similar high throughput assay, can be further evaluated using invitro procedures described herein and in PCT Publication WO/2008/008358(e.g., K_(cat) and K_(m) values, stability at 37°, melting temperature(T_(m)), endotoxin levels, ability to degrade substrate in plasma).Protein-compound compositions exhibiting thermostability over theprotein itself can be further evaluated when appropriate using in vivoprocedures described herein and in PCT Publication WO/2008/008358 (e.g.,potency, duration of action, effects with repeated dosing, and/orimmunological evaluation).

Thus, for these screening methods, the protein is preferably an enzyme.More preferably, the protein is a protease. Even more preferably, theprotein is an esterase. Most preferably, the protein is a cocaineesterase (CocE), having an amino acid sequence at least 80% identical toSEQ ID NO:1 and is capable of specifically catalyzing the cleavage ofcocaine into ecgonine methyl ester and benzoic acid. Preferably, theCocE has an amino acid sequence at least 90%, more preferably, 95%, evenmore preferably 99% identical to SEQ ID NO:1. In some preferredembodiments, the CocE has an amino acid sequence identical to SEQ IDNO:1.

Since it is desirable that the thermostabilizing compound is effectiveat a low concentration, it is preferable in these screening methods thatthe compound is present in the composition at a concentration of lessthan about 1 mM. More preferably, the compound is present in thecomposition at a concentration of less than about 0.1 mM. Mostpreferably, the compound is present in the composition at aconcentration of less than about 0.025 mM.

Preferably in these screening methods the thermostability is measured bymeasuring protein function at a low temperature and a high temperaturein the presence and absence of the compound, where the low temperatureis near the optimum temperature for protein function and where theprotein is less stable at the high temperature than at the lowtemperature. Preferably, the high temperature is about 37° C.,particularly when the protein is a CocE. However, for other enzymes, thehigh temperature can be greater than about 40° C., greater than about50° C., greater than about 60° C., greater than about 70° C., greaterthan about 80° C., greater than about 90° C., greater than about 95° C.,or even greater than about 98° C., e.g., with thermostable polymerasesfor PCR.

Any aspect of protein function can be measured to determinethermostability with and without the compound. Preferred examples ofprotein function for this purpose is enzyme activity and ligand binding.

The application is also directed to the use of the above compositionsfor the manufacture of a medicament for the treatment of acocaine-induced condition. Preferably, the cocaine-induced condition iscocaine overdose, cocaine toxicity, cocaine addiction, or cocainedependence. Most preferably, the cocaine-induced condition is cocaineoverdose.

The CocE for these uses can be a thermostable mutant of a wild-type CocEhaving the amino acid sequence of SEQ ID NO:1. Preferred such mutantshave the amino acid sequence of SEQ ID NO:1 except for the substitutionT172R, S159A, N197K, L169K, F189K, G173Q, or T172R/G173Q. In anotherthermostable mutant, the CocE has the amino acid sequence of SEQ ID NO:1except for the substitution L169K/G173Q. CocE mutants having more thanone mutation, preferably more than one thermostabilizing mutation, arealso useful for these treatment methods.

These uses are not limited to the utilization of any particularthermostabilizing compound. Preferably, the compound is

More preferably, the compound is

In other preferred embodiments, the compound is

The compound can also be

The compound can additionally be

Additionally, the compound can be

Further, the compound can be

The compound can also be

In these uses, the CocE can be combined with more than onethermostabilizing compound, for example one of the compounds describedabove, or with any other compound.

The application is additionally directed to the use of the abovecompositions for the treatment of a cocaine-induced condition.Preferably, the cocaine-induced condition is cocaine overdose, cocainetoxicity, cocaine addiction, or cocaine dependence. Most preferably, thecocaine-induced condition is cocaine overdose.

The CocE for these uses can be a thermostable mutant of a wild-type CocEhaving the amino acid sequence of SEQ ID NO:1. Preferred such mutantshave the amino acid sequence of SEQ ID NO:1 except for the substitutionT172R, S159A, N197K, L169K, F189K, G173Q, or T172R/G173Q. In anotherthermostable mutant, the CocE has the amino acid sequence of SEQ ID NO:1except for the substitution L169K/G173Q. CocE mutants having more thanone mutation, preferably more than one thermostabilizing mutation, arealso useful for these treatment methods.

These uses are not limited to the utilization of any particularthermostabilizing compound. Preferably, the compound is

More preferably, the compound is

In other preferred embodiments, the compound is

The compound can also be

The compound can additionally be

Additionally, the compound can be

Further, the compound can be

The compound can also be

In these uses, the CocE can be combined with more than onethermostabilizing compound, for example one of the compounds describedabove, or with any other compound.

The application is additionally directed to an isolated nucleic acidencoding a CocE polypeptide comprising an amino acid sequence that hasat least 85% sequence identity with the polypeptide of SEQ ID NO:1. Inthese embodiments, the encoded CocE polypeptide has (a) thesubstitutions L169K and G173Q, and (b) esterase activity with increasedthermostability at 37° C. as compared to wild-type CocE. See Example 5,establishing that this polypeptide has a half life of about 72 hours at37° C., which is more than 300× longer than the wild-type enzyme havingthe sequence of SEQ ID NO:1. Preferably, the amino acid sequence has atleast 90% sequence identity with the polypeptide of SEQ ID NO:1. Morepreferably, the amino acid sequence has at least 95% sequence identitywith the polypeptide of SEQ ID NO:1. Even more preferably, the aminoacid sequence has at least 99% sequence identity with the polypeptide ofSEQ ID NO:1. In the most preferred embodiments, the nucleic acid encodesa CocE polypeptide that has the sequence of SEQ ID NO:1 except for thesubstitutions L169K and G173Q.

The application is also directed to CocE polypeptides encoded by any ofthe above nucleic acids encoding a CocE polypeptide having the L169K andG173Q substitutions. In some embodiments, the CocE polypeptide is in apharmaceutically acceptable carrier.

In additional embodiments, the application is directed to a method oftreating a mammal undergoing a cocaine-induced condition. The methodcomprises administering the above-described composition comprising theCocE polypeptide having L169K and G173Q substitutions to the mammal in amanner sufficient to reduce the effects of the cocaine-induced conditionon the mammal.

Further, the application is directed to the use of the above-describedcomposition comprising the CocE polypeptide having L169K and G173Qsubstitutions for the manufacture of a medicament for the treatment of acocaine-induced condition.

The application is additionally directed to the use of theabove-described composition comprising the CocE polypeptide having L169Kand G173Q for the treatment of a cocaine-induced condition.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims, which follow the examples.

Example 1 Compounds that Thermostabilize CocE

Cocaine esterase is a bacterially expressed protein that catalyzes thecleavage of cocaine into two inactive byproducts: ecgonine methyl esterand benzoic acid. The protein could theoretically be used in vivo fortreatment of cocaine overdose and addiction, however the wild-typeprotein is not stable at 37° C.

Analysis of CocE action on cocaine cleavage is performed by utilizingthe spectroscopic properties of cocaine, which maximally absorbs lightat a 240 nm wavelength. CocE activity is measured by monitoring for adecrease in signal at A240, using various concentrations of cocaine, anddetermining the initial rate of the decrease. From these values theV_(max) of the enzyme can be determined. By pre-incubating the enzyme at37° C. for various times before monitoring for activity, the half lifeof CocE at 37° C. can be calculated.

In an ongoing effort to improve CocE thermostability, several mutants ofthe protein have been made and tested for in vitro half life. Subsequentanalysis using gel electrophoresis showed that under native conditionsthe proteins could be observed to aggregate after preincubation at 37°C. for various times (PCT Publication WO/2008/008358). The disappearanceof the initial product can be measured by densitometry analysis, andthis analysis supported the spectroscopic data that the mutants had animproved in vitro half-life over the WT and S167A mutant.

While pre-incubation at various times indicates that WT CocE had a shorthalf life at 37° C. (5 minutes), it was also observed that ifspectrophotometric measurements were run at 37° C., then CocE WT wouldcontinue to cleave at a linear rate for more than 60 minutes (FIG. 2,middle) or until the cocaine substrate had been exhausted (FIG. 2,bottom). This suggested that the WT enzyme was being stabilized in thepresence of cocaine or it's byproducts.

CocE is also able to cleave another substrate, 4-nitrophenyl acetate(FIG. 3). Cleavage is monitored by detection of the appearance of the4-nitrophenol reaction product, which absorbs at 400 nm. Analysis ofcleavage of this substrate at 37° C. shows the product is initiallyproduced quickly, but the reaction slows over time (FIG. 3, middle andbottom). This indicates that the 4-nitrophenyl acetate and products areprobably not stabilizing the enzyme, or at least not to the degree thatthe cocaine reaction is.

To analyze the stabilization of CocE WT by substrates and products, theability of each compound to inhibit formation of CocE protein aggregateswas tested after incubation for 1 hour at 37° C. Both cocaine andbenzoic acid inhibited the formation of aggregates at certainconcentrations, but ecognine methyl ester and sodium acetate could not(FIG. 4).

Further spectrophotometric analysis of cocaine stabilization wasperformed, by analyzing the ability of CocE to cleave 4-nitrophenylacetate, after preincubation at 37° C. for various times, in thepresence of various concentrations of cocaine. Cocaine was found to be ainhibitor of 4-nitrophenyl acetate cleavage at higher concentrations(62.5 and 125 μM)(FIG. 5). Concentrations lower than this were not foundto be stabilizing at 37° C. However, preincubation of CocE at the higherconcentrations for various times at 37° C. indicated the enzyme wasstabilized enough to be able to cleave the 4-nitrophenyl substrate (FIG.5).

Kinetic spectrophotometric analysis of benzoic acid, ecgonine methylester and sodium acetate stabilization of CocE did not reveal anystabilization below 125 μM (FIG. 6). However benzoic acid was found toboth inhibit and stabilize at higher concentrations.

To further study CocE stabilization with small molecules, otherinhibitors of the CocE cleavage reaction were considered. Phenylboronicacid, an irreversible inhibitor of CocE, which was able to stabilizeCocE WT aggregation at 37° C. for 1 hour, with a 50% stabilizationconcentration of approximately 0.2 μM (FIG. 7).

The data described above indicates that wild-type CocE was able to bestabilized by the addition of substrates, products or inhibitors of theenzyme. However all of these small molecules inhibited enzyme activityto certain degrees. It was evaluated whether there might exist a smallmolecule that could prevent CocE aggregation, without inhibiting enzymeactivity. Such a molecule could be used as a stabilizer in vitro, e.g.,during enzyme preparation, and in vivo. An assay was designed to screena library of 20,000 small molecules for stabilization of CocE. In thatassay, the enzyme is mixed with the compounds in a 96-well plate format,and then incubated at 37° C. for one hour. The controls for the assayare usually the compound diluent (e.g., DMSO), unheated enzyme, andenzyme mixed with 2000 μM benzoic acid. After 37° C. incubation, theenzyme/compound mixtures are tested for the ability to cleave4-nitrophenyl acetate (FIG. 8). Only compounds stabilized would be ableto cleave after this incubation period.

Forty compounds were first tested. The compounds were assayed induplicate with appropriate controls. A duplicate plate (without enzyme)was performed as a negative control, to check for compounds able tocause an increase in 400 nm absorbance in the absence of CocE. Severalcompounds were found to also increase absorbance at 400 nm. Some wereable to do this in the absence of CocE. These were discarded as falsepositives.

FIG. 9 is a plot of the initial rate of 4-nitrophenyl acetate cleavagefor all compounds, after the background “no enzyme” cleavage wassubtracted. Compounds with significant activity (2 standard deviationsabove DMSO only controls) are marked with an asterisk and the chemicalstructures of those compounds are shown.

The most effective compound in the assay was compound 6031818 (FIG. 10).That compound is a weak inhibitor of 4-nitrophenyl acetate cleavage (16μM; FIG. 10, left graph), and does not inhibit cocaine cleavage at all(FIG. 10, right graph).

The stabilization assay in the presence of high enzyme concentrationsindicated that enzyme activity begins to drop after 60 minutes at 37° C.(FIG. 11). After that time, 6031818 was able to stabilize the half lifeof the wild-type enzyme, such that at 20 μM the half life increases from12-14 minutes to 60-70 minutes.

Another effective compound, 6169221, had a very similar structure to6031818. This compound also did not inhibit 4-nitrophenyl acetate orcocaine substrate cleavage (FIG. 12). The 6169221 compound was onlyweakly able to stabilize the enzyme, increasing the half life from 7-12minutes to 13-17 minutes.

Another thermostabilizing compound is 5804238. That compound also didnot inhibit either 4-nitrophenyl acetate or cocaine cleavage (FIG. 14).5804238 was weakly able to stabilize the enzyme, increasing the halflife from 9-10 minutes to 22-25 minutes (FIG. 15).

Example 2 Circular Dichroism of Wild Type and Mutant CocE

Further characterization of the stability of wild type (WT) CocE andthermostable mutants was performed through circular dichroism, whichdetects small changes in protein conformation and structure. Analysis byrepeated CD measurements at increasing temperatures allows analysis ofthe thermodynamics and melting temperature (T_(M)) of the protein, solong as the melting is reversible (i.e. the protein resumes it'soriginal conformation upon cooling). Unfortunately, the melting of CocEWT is not reversible, so true thermodynamics cannot be determined.However CD is still of value in determining the temperature tounfolding. In these assays, CocE WT (FIG. 16, top) melts atapproximately 39° C., whereas the T172R mutant (FIG. 16, bottom) meltsat ˜42° C., showing the thermostability of this mutant conferred at 37°C. is due to a melting temperature 2-3 degrees higher than the WT.

WT CocE in the presence of excess benzoic acid (FIG. 17, top) increasedthe melting temperature of CocE to 53° C., a full 10 degrees higher thanthe T172R mutant. The benzoic acid molecule at this concentration wasslightly spectroscopic, affecting the spectra of the protein, but notthe melt. Analysis of WT CocE in the presence of 5× molarityphenylboronic acid (FIG. 17, bottom) increased the melting temperatureto 73° C., that is, more than 30 degrees higher than the WT and T172Rmutant.

CD analysis of another CocE mutant, L169K, established a ˜65° C. meltingtemperature alone, and ˜85° C. melting temperature in the presence ofphenylboronic acid (FIG. 18), i.e. phenylboronic acid conferred anadditional 20° C. melting to the already high original meltingtemperature for L169K.

CD analysis of CocE WT in the presence of 6031818 established a meltingtemperature of ˜42° C., similar to the T172R mutant (FIG. 19).

Example 3 Analysis of Thermostability of Various Compounds withWild-Type and a Mutant CocE

Wild-type CocE or CocE mutant L169K was incubated in the presence ofvarious small molecules at 37° C. for various time points, then run onnon-denaturing gels. The results are shown in FIG. 20. Spot densitometrywas used to analyze the gels to determine the half-life of the CocEenzymes under these conditions. See FIG. 21. Phenylboronic acidincreased the half-life of the CocE the most, followed by benzoic acid.Compound 6041818 increased the half-life of the wild-type enzyme byabout 50%.

Example 4 Identification of CocE Thermostable Mutants by Structure-BasedAnalysis Example Summary

Despite advances in the development of therapeutics that target thedopamine transporter, the identification of therapeutics that combatcocaine abuse and overdose have been less fruitful. More classicalapproaches to therapeutic development against cocaine abuse and overdosehave inherent challenges in that competitive and allosteric inhibitorsof cocaine binding to the transporter exhibit similar behavioral effectsof cocaine: inhibition of dopamine uptake. The use of cocaine esterasehas been developed as a protective therapy against cocaine-inducelethality. The acceleration of enzyme-mediated digestion of systemiccocaine by exogenously added cocaine esterase represents a significantparadigm shift in cocaine abuse therapy. Here the design and generationof significantly more stable enzyme preparations using computationalapproaches is reported. Evidence from both in vitro and in vivo studiesis provided indicating that the modified enzyme displays a prolongedhalf-life (up to 30-fold) and improved thermostability than thewild-type enzyme. Moreover x-ray crystallographic evidence has beenobtained that provide a structural rationale for the improved enzymestability.

INTRODUCTION

Structure-based and computational approaches were utilized to generatemutants of CocE with increased stability at 37° C. The crystal structureof CocE (Larsen et al., 2002) and a combination of molecular modeling,energy minimizations, and molecular dynamics (MD) simulations with theRosettaDesign program (Kirjegian et al., 2005; Kuhlman and Baker, 2000)and AMBER program (Case et al., 2004) were used. The expressed andpurified mutants were assessed for their improved intrinsic stabilityusing in vitro assays. Importantly, three out of 36 predictedsubstitutions exhibited a dramatic improvement in the half-life of theenzyme as assessed by in vitro assays and by the in vivo protectionagainst cocaine-induced lethality. X-ray crystal structures of thesemutants were determined in order to investigate their structural basisfor improved thermal stability. In each case, the substitutions increaseinterdomain contacts of the enzyme. The dramatic improvement instability of mutant CocE in vivo illustrates the promise of both thisexperimental approach, and the use of CocE in cocaine abuse andaddiction.

Materials and Methods

Materials.

Cocaine was purchased from Mallinckrodt Inc., St. Louis, Mo. All otherreagents are of analytical grade and were obtained from FisherBiosciences and Sigma-Aldrich Corp.

Design of Thermostable Mutations.

Based on the X-ray crystal structure (PDB code 1JU3) of the bacterialcocaine esterase (cocE) (Larson et al., 2002), a complete 3D model ofCocE bound to (−)-cocaine was built using energy minimizations andmolecular dynamics (MD) simulations encumbered within the AMBER program(Case et al., 2004). To increase the thermostability of CocE, acomputational method implemented in RosettaDesign program (Kuhlman andBaker, 2000) was used, which was capable of predicting thermostabilizingmutations within a given fold while minimizing any shift in the backbonethat might structurally disrupt the active site structure or quench itsflexibility. The method implemented in the RosettaDesign program uses anenergy function for evaluating the fitness of a particular sequence fora given fold and a Monte Carlo search algorithm for sampling sequencespace. The same method has successfully been used by other researchersto increase thermostability of an enzyme with no reduction in catalyticefficiency (Korkegian et al., 2005; Kuhlman and Baker, 2000). Thecomputational modeling using the RosettaDesign program has allowed theprediction of a set of mutations that can potentially lower the energyand, therefore, increase the higher thermostability of CocE. As thefirst round of the rational design, the computation was simplified byonly considering the possible mutations on the amino acid residues thathave a distance of 6-25 Å from cocaine.

Site Directed Mutagenesis.

Point mutations using CocE cDNA cloned in the bacterial expressionvector, pET-22b(+), as a template were generated using a modifiedQuickChange (Stratagene) mutagenesis protocol and single oligonucleotideprimers. For generation of double mutants, cDNAs with single pointmutations were used as templates for a second round of mutagenesis. Allmutants were sequenced in both directions over the entire coding region.Wild-type and CocE mutants were expressed as C-terminally-6× His-taggedproteins in E. coli BL-21 Gold (DE3) cells grown at 37° C. Proteinexpression was induced with 1 mM isopropyl-β-thiogalactopyranoside(IPTG, Fisher) for 12 hours at 18° C.

Purification of Cocaine Esterase and Mutants.

Cells were pelleted, resuspended in 50 mM Tris pH 8.0, 150 mM NaCl withprotease inhibitors (3 μg/ml each of leupeptin and lima bean or soybeantrypsin inhibitor) and lysed using a French press (Thermo FisherScientific Corp, USA). Wild-type or mutant CocE was enriched using Talonmetal affinity chromatography (Clontech Laboratories, Inc, Mountain ViewCalif.), purified using anion-exchange (Q-Sepharose, GE Healthcare,Piscataway N.J.) chromatography. CocE was eluted from the Q-Sepharosecolumn with 150-450 mM NaCl linear gradient buffers containing 20 mMHepes pH 8.0, 2 mM MgCl₂, 1 mM EDTA and 1 mM DTT. The peak fractionswere pooled and concentrated by Centricon-30 (Millipore), snap frozen inliquid nitrogen and stored at −80° C.

Michaelis-Menten Kinetics of Cocaine Hydrolysis.

A spectrophotometric real-time assay of cocaine hydrolysis used tomonitor the hydrolysis of cocaine (Landry et al., 1993). The initialrates (of decay) were determined by following the change in theintrinsic absorbance of cocaine at 240 nm (6700 M¹ cm¹)(Xie et al.,1999) using a SpectraMax Plus 384 UV plate reader (Molecular Devices,Sunnyvale, Calif.) using SOFTmax Pro software (Version 3.1.2). Thereaction was initiated by adding 100 μL of a 2× enzyme solution (100 mMphosphate buffer, pH 7.4 and 300 mM NaCl) to 100 μL of a 2× cocainesolution (50 μg/mL enzyme, 100 mM Phosphate Buffer, pH 7.4 and 300 mMNaCl). All assays were performed with 100 μM DTT unless indicatedotherwise. Final cocaine concentrations were as follows: 125, 62.5,31.25, 15.63, 7.81, 3.91, 1.95, and 0.977 μM. V_(max) and K_(m) valueswere calculated using Prism (GraphPad Software, San Diego). Forstability measurements, wild type and mutant enzymes were diluted to 2×concentration and incubated at 37° C. for the times indicated. At theend of each time point, an aliquot was removed and kinetic behavior wasobserved as outlined above.

In Vivo Protection Against Cocaine Lethality.

Male NIH-Swiss mice (25-32 g) were obtained from Harlan Inc.(Indianapolis, Ind.) and were housed in groups of 6 mice per cage. Allmice were allowed ad libitum access to food and water, and weremaintained on a 12-h light-dark cycle with lights on at 06.30 AM in aroom kept at a temperature of 21-22° C. Experiments were performed inaccordance with the Guide for the Care and Use of Laboratory Animals asadopted and promulgated by the National Institutes of Health. Theexperimental protocols were approved by the University Committee on theUse and Care of Animals at the University of Michigan.

Cocaine-induced toxicity was characterized by the occurrence oflethality, as defined by the cessation of observed movement andrespiration. Mice were placed individually in Plexiglas observationchambers (16×28×20 cm high) to be habituated for 15 min before drugadministration. Following intra-peritoneal (i.p.) cocaineadministration, the mouse was immediately placed individually in thesame chamber for observation. The presence or absence of lethality wasrecorded for 60 min following cocaine administration. The mouse wasplaced in a small restraint chamber (Outer tube diameter: 30 mm; Innertube diameter: 24 mm) that left the tail exposed. The tail was cleansedwith an alcohol wipe and a 30G1/2 precision glide needle (FisherScientific, Pittsburgh, Pa.) was inserted into one of the side veins forinfusion. The i.v. injection volume of CocE or CocE mutant was 0.2 mLper mouse. Sterile gauze and pressure were applied to the injection siteto staunch the bleeding.

The potency of CocE mutants to protect against cocaine-induced toxicitywas assessed following i.v. enzyme administration (0.3 or 1 mg) 1 minprior to administration of several doses of i.p. cocaine (180, 320, 560,and 1000 mg/kg, n=8/dose). Dose-response curves of cocaine-inducedlethality in the absence or presence of a single dose of the enzyme weredetermined to demonstrate the in vivo protective effects of CocEmutants.

The duration of protection against cocaine toxicity provided by CocE andCocE mutants was assessed through monitoring lethality following i.v.enzyme administration (0.1, 0.3, and 1 mg) prior to i.p. cocaine (LD₁₀₀,180 mg/kg). Lethality was monitored following injection at 1, 5, 10, or30 min, or 1, 2, 3, 4, 5 hours after enzyme administration. Eachtreatment used 8 mice to assess the percent of lethality (i.e.,protection) in mice pretreated with a single dose of an esterase at asingle time point.

In the potency study, a group LD₅₀ value was calculated by least-squaresregression using the portion of the dose-response curve spanning 50%occurrence of lethality. These values were used to compare the degree ofrightward shifts of cocaine's dose-response curve in the absence orpresence of the enzyme pretreatment. In the time course study, a timepoint for duration of protection (i.e., 50% of lethality) was estimatedby using each time course curve crossing 50% occurrence of lethality.

Cocaine hydrochloride (Mallinckrodt Inc., St. Louis, Mo.) was dissolvedin sterile water and was administered intraperitoneally at a volume of0.01 mL/g. CocE or CocE mutant was diluted to different concentrationsin phosphate-buffered saline and administered intravenously at a volumeof 0.2 mL/mouse.

Crystallization and Structure Determination.

Crystals were grown by the hanging drop vapor diffusion method aspreviously described (Larsen et al., 2002). For harvesting, 2 μL ofcryoprotectant (5 mM Tris pH 7.0, 1.5 M ammonium sulfate, 10 mM HEPES pH7.5, 2 mM MgCl₂, 1 mM EDTA, 825 mM NaCl, 25% glycerol and 1 mM DTT whereindicated) were added to each hanging drop, and then crystals weretransferred to 100% cryoprotectant and flash-frozen in liquid nitrogen.Crystals were harvested within 3 days after tray set-up.

Diffraction intensities were collected at the Advanced Photon Source atbeamlines supported by GM/CA- and LS-CAT, and then reduced and scaledusing HKL2000 (Otwinowski et al., 1997). Initial phases were fromstraightforward molecular replacement using previously publishedstructures of CocE (Larsen et al., 2002). REFMAC5 was used for maximumlikelihood refinement and model-building and water addition wereperformed with O and COOT. Unambiguous density was present for allmutated side-chains. Twenty-three total datasets were collected withmultiple data sets for each crystal type so the behavior of the H2-H3loop could be compared. Figures were generated with PyMol[http://www.pymol.org]. Coordinates and structure factors are depositedin the PDB under accession codes 2QAY (T172R), 2QAX (G173Q), 2QAW(T172R/G173Q), 2QAV (L169K), 2QAT (wild-type without ligand) and 2QAU(wild-type with DTT adduct).

Results

Design of Thermostable Mutations.

Cocaine esterase is contains three distinct domains. Domain I (residues1-144 and 241-354) compose the canonical α/β-hydrolase fold. Domain II(residues 145-240) is a series of 7 α-helices inserted between strandsβ6-β7 of Domain I. Domain III (355-574) primarily consists of β-sheetsand comprises an overall jelly roll-like topology (FIG. 22A).Computational studies were performed, including MD simulation andsubsequent energetic analysis to identify substitutions within the 6-25Å shell surrounding the active site that would stabilize CocE. Thisstructure-and-mechanism-based design of the CocE mutants combined theuse of energy minimizations and MD simulations using AMBER (Case et al.,2004) and further modeling studies using the Rosetta Design program(Kuhlman and Baker, 2000). Although CocE is a dimer in solution and incrystals, the modeling was performed with a monomer. The data summarizedin Table 1 suggest that the following mutations could thermodynamicallystabilize the CocE structure: R41I, N42V, K46A, S56G, T74S, F84Y, L119A,V121D, T122A, Q123E, S140A, L146P, A149S, Y152H, S159A, L163V, V160A,S167A, T172R, G173Q, F189L, A193D, A194R, I218L, W220A, V225I, T254R,V262L, S265A, W285T, A310D, C477T, L508G, K531A, Y532F, and D533S. Thepositions of the mutations on the CocE structure are shown in FIG. 22A.Each of these single mutations is predicted to stabilize the CocEstructure by 2.1 to 4.5 kcal/mol, suggesting that the half-life time ofthe protein should become about 30 to 1000-fold longer at roomtemperature (Table 1). To test these predictions, cDNAs encoding themutations in CocE were expressed in E. coli and the resulting proteinscharacterized by kinetic and stability assays. Out of the 36 mutantstested, three mutations that clustered around helix 2 of Domain IIappeared to improve the enzyme stability at 37° C. without significantreduction in catalytic efficiency, as described below.

TABLE 1 Mutant CocE displaying enhanced stability following incubationat 37° C. Mutant Stability @ 37 (t_(1/2)) T122A No Q123E No S159A NoS140A No S167A/W52L No T172R ~46 min V121D No L163V No F189A NoF189A/T172R ~40 min (Similar to T172R) C107S No W220A No F189L No A193DNo T172R/A193D ~40 min (to T172R) G173Q ~25 min T254R No N42V NoT172R/G173Q ~326 min G171Q/T172R/G173Q No G171A No G173A Nowt-I175-G-D185 No wt-T176-G-G-D185 No T172R/G173Q-I175-G-D185T172R/G173Q-I175-G-G-A186 ~75 min T172R/G173Q-T176-G-G-D185 ~75 minS177Q No D45R No F47R No L169K ~274 min L174R No A181K No S179R No F189K25 min V190K No A194K No R182K No τ_(1/2) were determined bypreincubation of the enzyme at 37° C. for varying times. Activitymeasurements were determined at RT (25° C.). Mutant enzymes with τ_(1/2)of 12 min (i.e. the τ_(1/2) of wildtype [wt] CocE) or less wereconsidered not thermally stable. This Table is also in WO/2008/008358.

In Vitro Kinetic Assays.

To assess the enzymatic activity of wt and mutant CocE, the hydrolysisof cocaine was directly measured at 37° C. using a spectrophotometicassay (Xie et al., 1999). Initial rates were then used to determineMichaelis-Menten parameters. To assess thermal stability, enzymepreparations were preincubated at 37° C. for various lengths of time,prior to measurement of residual activities (Table 1 and FIG. 23).Inactivation at 37° C. occurs in a time-dependent manner decay. In theabsence of DTT, pre-incubation of wt-CocE at 37° C. decreases enzymeactivity exponentially with a half-time of inactivation, τ_(inact),approximately 25 min. Three out of 36 of the predicted mutants increasedthe enzymatic stability of CocE: T172R (τ_(inact)=46 min), G173Q(τ_(inact)=35 min; data not shown), and L169K (τ_(inact)˜274 min). WhileT172R and G173Q mutants did not appear to deleteriously effect theenzyme's catalytic efficiency (k_(cat)˜1×10⁸ and 2×10⁸, respectively),the k_(cat) of L169K was diminished, largely as a result of ˜5 foldincrease in its K_(m) for cocaine esterase (Table 2). Interestingly, themutants that appear to display significant stability at 37° C. allreside on helix 2 of Domain II (FIG. 22B). Domain II is also locatednear to the active site and may, at least in the case of L169K, accountfor the effects on k_(cat). Also noteworthy is the observation thatincubating the enzyme with DTT appears to accelerate the decay for thewild-type enzyme, but not T172R/G173Q and L169K (FIG. 24). It was alsodetermined that DTT can inhibit cocaine hydrolysis with a K_(i)˜380 μM(FIG. 25) in a manner that appears to be mixed competitive andnon-competitive (not shown). No effect of DTT was observed whencombining the mutations appears to further enhance enzyme stability at37° C. (not shown).

TABLE 2 Kinetic behavior of wild-type and redesigned CocE mutants.Catalytic K_(cat()(mol · Efficiency, Enzyme t_(1/2) (min) s⁻¹ · mol⁻¹)K_(m)(M) K_(cat)/K_(m) (s⁻¹) wt-CocE 12.2 2323 0.021 1.11E+08 T172R 46.82502 0.024 1.05E+08 T172R/G173Q 326 2247 0.024 9.40E+07 L169K 274 31040.105 2.90E+07 The metabolism of cocaine by purified preparations ofwt-CocE, T172R, T172R/G173Q or L169K was measured as described in theMethods Section. The Michaelis constant, K_(M), and K_(cat) wereestimated using Prism (Graphpad, San Diego, CA).

The activity of T172R/G173Q, while still sensitive to incubation at 37°C. displays an enhanced stability and decays τ_(inact)˜326 min. However,the observed catalytic activity plateaus to approximately 35% of itsmaximal catalytic rate (i.e. at t=10 hours). This inactivation profilewas qualitatively and quantitatively different than the behavior of theT172R and G173Q single mutants and wt-CocE, but similar to that ofL169K. Surprisingly, the triple mutant (L169K/G173Q/T172R) did notdisplay an enhanced stabilization (data not shown).

To test whether the improved enzymatic stability at 37° C. representsimproved thermal stability of the protein fold, the capacity of the CocEat progressively higher temperatures were assessed (FIG. 26). Theactivity of wt CocE plummets precipitously after 30 min at approximately30-35° C. Both L169K and T172R/G173Q each are inactivated at a highertemperature (40-45° C.). Circular dichroism analysis (near UV analysis)at varying temperatures, comparing wt-CocE and T172R/G173Q have meltingtemperatures in concordance with the loss of catalytic activity.

In Vivo Assays.

Pretreatment with wt-CocE, L169K, T172R, or T172R-G173Q 1 min prior tococaine administration protected mice against cocaine-induced lethality(FIG. 27). The enzyme protection (at 0.3 mg, or 9 mg/kg) altered theLD₅₀ value of cocaine of 100 mg/kg, for the vehicle-treated group, to560 and 670 mg/kg for wt-CocE, T172R, or T172R-G173Q (FIG. 28). L169Kwas slightly less effective and required larger doses (1 mg, or 30mg/kg) to produce a similar 6-7-fold rightward shift in the cocainedose-response curve, consistent with the decreased catalytic efficiencyobserved in in vitro assays.

Although pretreatment (greater than 30 min) with low doses of eitherenzyme (0.1 mg) were ineffective against the lethal effects of cocaine,larger doses (0.3 mg and 1 mg) appeared to be effective, the durationsof which were dependent on the mutation (FIG. 28 and summarized in FIG.29). At the largest doses tested (1 mg) the enzyme pretreatment timenecessary to protect to 50% lethality, LT₅₀, for wt-CocE wasapproximately 14 min. Considerably longer LT₅₀s were observed for T172R(LT₅₀˜1.8 hr), L169K (LT₅₀˜3.3 hr) and T172R-G173Q (LT₅₀˜4.5 h),consistent with the in vitro data.

Structural Analysis of Stabilizing Mutants.

High-resolution X-ray crystal structures of wt-CocE (1.5 Å), as well asthermal-stable mutants L169K (1.6 Å), T172R (2.0 Å), G173Q (2.5 Å), andT172R/G173Q (2.0 Å) were determined. FIG. 30 summarizes some of theresults. Delineation of the structure of unliganded wt-CocE has notpreviously been reported and was necessary for comparison in our study.

The structures of L169K, T172R, G173Q, and T172R/G173Q all showwell-ordered density for their mutated side-chains (FIG. 31). In eachcase, the substitutions appear to increase the number of contacts/buriedsurface area between domains of CocE. Elongation of the side group alkylchain and the addition of a guanidinium moiety through substitution ofarginine in T172R generates both van der Waals contacts with thearomatic ring of F189 in helix H3 and a hydrogen bond between theguanidinium moiety to the backbone oxygen of F189 (FIG. 31A, B). Theguanidinium side chain also packs against the I205 side chain donated bythe dimer-related subunit. The side chain of G173Q forms a trans-domainhydrogen bond with the backbone carbonyl of P43 in domain I, and van derWaals contacts with Y44, whose hydroxyl contributes to the oxyanion holeof the active site (FIG. 31C,D). The L169K substitution also formscontacts with the phenyl ring of Y44 in domain I (FIG. 31E,F). Thelonger side chain of lysine could impinge upon the binding site of thetropane ring of cocaine, perhaps accounting for the higher K_(m)exhibited by this mutant.

In previously reported structures (1JU3 and 1JU4) and in ours, weobserve multiple distinct conformations for a region encompassing theC-terminus of the H2 and the H2-H3 loop (residues 171-183, asillustrated in FIG. 32). These two conformations are likely to be inequilibrium while in solution, but the population of these two statesappears to be influenced by the mutations within the H2. Because thestabilizing mutants are found in the H2 helix, it was hypothesized thatthe mutants may help reduce the conformational flexibility of thisregion, and thus thermally protect the fold of the enzyme. For thisanalysis, the “out” conformation of this region was defined as thattypified by the 1JU3 phenyl boronic acid adduct crystal structure,wherein this region bends away from the H5-H6 helices of domain I by upto 3.3 Å compared to the “in” conformation, typified by the 1JU4 benzoicacid crystal structure. A third conformation of this loop region wasalso observed, which is similar to that of 1JU4 except that residues178-181 have a unique conformation.

The apparent global flexibility of H2-H3 mandates distinct side chainconformations within each helix. For example, in the ‘out’ conformation,I175 moves towards H3, forcing F189 out of the hydrophobic interfacebetween H2 and H3, while the ‘in’ conformation allows F189 to be eitherin or out of this interface.

The structure of the T172R mutant reveals a tendency of F189 to adopt an“out” position via the close contacts of the R172 and F189 side chains,a tendency that is more prevalent in the T172R/G173Q mutant. These datawould suggest that locking the planar conformation of F189 maycontribute toward the thermal stabilizing effects of the T172Rsubstitution. Substitution of alanine for phenylalanine at 189 does notreveal any enhancement or diminution of thermal stability in the T172Ror wildtype background. Thus, the interactions of R172 with the H3 helixitself or perhaps with the dimer related subunit appear responsible forthe enhanced stability.

Formation of a DTT-Carbonate Adduct in the Active Site of CocE.

Complicating these studies was exceptionally strong density in theactive site that appeared to correspond to a covalent adduct with thecatalytic serine of CocE (Ser117). Such density was not previouslyreported (FIG. 33) (Larsen et al., 2002). The electron densitycorresponded to a five membered ring with two substituent arms.Anomalous difference Fourier maps confirmed the presence of sulfur ineach of the arms, and 2F_(o)—F_(c) omit maps contoured at differentlevels identified the oxygen atoms in the adduct (FIG. 33). Thus, it wasconcluded that the density corresponds to DTT, which was included in thecrystallization and harvesting solutions (at 10 mM), that was reactedwith bicarbonate in the active site. The 2-oxo-dioxolane ring appearstrapped as a tetrahedral intermediate dead-end complex, with one of thetetrahedral oxygens occupying the oxyanion hole forming the adduct,2-oxo-dioxolane butyryl carbonate, or DBC. In the highest resolutionstructure (L169K), the carbon presumably donated by carbonate in thedioxolane ring is ˜1.6 Å (distance was not restrained in refinement ofthe high resolution structures) from the S117 γ-oxygen, the oxygen inthe oxyanion hole, and 1.4-1.5 Å from the oxygen in the oxyanion holeand the two oxygen atoms donated by DTT, most consistent with covalentbounds. The electron density of this tetrahedral carbon is weaker thanthat of the other carbons in the DTT ligand, suggesting electronwithdrawal. The 2-oxo-dioxolane adduct adopts a similar conformation tothe 2-phenylboronate adduct with CocE (Larsen et al., 2002), except thatthe tetrahedral center is rotated.

To confirm that formation of this adduct complex was not a consequenceof our stabilizing mutants, the wt-CocE structure was determined in thepresence and absence of DTT, and the structures of stabilizing mutantsof CocE were also determined in the absence of DTT. In all cases whereDTT was co-crystallized with CocE, the adduct was observed, and theposition of the H2-H3 insert was essentially the same with or withoutDTT. Under the in vitro conditions tested here, i.e. relatively shortincubation times with DTT (<60 min), DTT inhibits CocE activitycompetitively with substrate. Note that crystal growth conditions areconsiderably different with incubation times in the days to weeks, atime scale that could conceivably allow for the formation of the DBCadduct. Adduct formation should also display markedly differentinhibition patterns and appear as a non-competitive inhibitor. Crystalsgrown without DTT, or grown with DTT and subsequently soaked in thecocaine analog atropine which appeared to displace the adduct, insteadshowed a water molecule near S117 and high B-factors for active siteresidues including S117 and H287 of the catalytic triad.

Discussion

To date, CocE is the most efficient catalyst for hydrolyzing cocaine andfor decreasing cocaine levels in vivo and to protect againstcocaine-induced lethality in mice and rats (Turner et al., 2002; Ko etal., 2007; Cooper et al., 2006; Garrera et al., 2005; Gasior et al.,2000; Daniels et al., 2006; WO/2008/08358). The effectiveness of this“antidote” for cocaine toxicity in rodents indicates that CocE is apotential therapeutic in humans. However, wt-CocE displays considerableinstability as its effective half-life in the blood stream is ˜10 min.In comparison, tetrameric BchE, remains in mice plasma for 16 hours andactive for up to 7 hour post injection (Duysen et al., 2002) whileanti-cocaine Ab, remains in mouse circulation for 8.1 days (Norman etal., 2007). Even so, the clinical potential of wt-CocE suggests that itsduration of protection is likely sufficient in acute overdose cases,such as those due to snorting or injection (Landry et al., 1993).

In cases involving massive overdoses, as is the case for “cocaine mules”wherein large amounts of cocaine will be released into the bloodstreamover a long period of time, a longer acting CocE is desired. The shorteffective plasma half-life of CocE therefore represents a major obstaclein developing this protein-based therapeutic for acute treatment ofcocaine-induced lethality and for chronic treatment of cocaine abuse.

Here, in vitro data is provided demonstrating that the relatively shorthalf-life in vivo may be a result of the thermal inactivation of CocEreadily observed in vitro. The thermal sensitivity of CocE may reflectthe fact that Rhodococcus sp., the microorganism which CocE was isolatedfrom, thrives in the soil beneath coca plants under moderatetemperatures around 20° C. (Mackay, 1886; Martin, 1952), much lower thanthe body temperature of rodents (37-38° C.).

Of the 36 mutants predicted computationally, three mutations displayedan enhanced thermal stability. A number of the mutants were not stableand could not be overexpressed and purified as a functional enzyme(Table 1). By combining two of the thermal stable point mutants, athermally quite stable mutant of CocE was created, G173Q/T172R, whichextends the in vitro τ_(inact) at 37° C. from 10 min to 4½ hours, orapproximately 27-fold. In vivo analysis of the mutants in rodents, as afunction of their capacity to protect against acute cocaine-inducedlethality, were in concordance with the in vitro assays.

The results of computational modeling studies were striking in thatseveral stable mutants were identified in an enzyme of 574 amino acidresidues. Unfortunately, the resolution of these methods were notsufficient enough to elucidate the precise mechanism underlying thethermal stabilizing effects of the mutants. In combination with x-raycrystallography, however, it was possible to ascertain a reasonablemodel to account for the enhance stability at an atomic resolution. Ingeneral the substitution of larger or charged residues such as glutamine(for glycine), lysine (for leucine) or arginine (for threonine), helpedto stabilize domain-domain interactions. The most thermally unstabledomain in the enzyme was identified as Domain II, which contains the H2and H3 helices.

The location of the thermally stable substitutions in the H2-H3 helices,and the structural heterogeneity of the H2-H3 loop itself, suggests thatthe H2-H3 helical region is inherently unstable and may ultimatelynucleate or at least contribute strongly to the aggregation or unfoldingof CocE. CocE orthologs from Listeria and Pseudomonas sp., both of whichare capable of surviving at 37° C., have significantly shorter H2 & H3helices and therefore a potentially more stable domain 2 (GenbankAccession codes ZP 01928677 and YP 660510, respectively). Truncation orcomplete removal of the loop between the H2 and H3 helices in CocE,however, inactivated the enzyme (see Table 1).

The presence of a DTT-carbonate adduct in the active site of CocE isapparently catalyzed by the enzyme. Other carbonates, such as propylenecarbonate (4-methyl-2-oxo-1,3-dioxolane), are reported to decompose intopropylene glycol and CO₂ in water. The reverse synthesis from DTT, avicinal diol, and CO₂ is thus conceivable. The DTT adduct appears to bebound covalently to the catalytic S117 much in the overall manner asphenyl boronic acid (Larsen et al., 2002), although its bond length of1.6 Å (as opposed to the expected distance of ˜1.43 Å) suggests partialcovalent character. The plane of the dioxolane ring of DBC overlaps theplane of the phenyl ring of both phenyl boronic acid- and benzoicacid-bound forms of CocE (Larsen et al., 2002). Because the conformationof the active site residues of wt-CocE bound to DBC differs very littlefrom the phenyl boronic acid-bound form, its possible that the adductwas not observed in the crystal structure by Larson et al. (2002) due tothe mutually exclusive binding of benzoic acid or phenyl boronate.

The crystal structure of CocE suggests that the enzyme exists as adimer, and preliminary studies suggest that the dimer is stable enoughat low concentrations to be resolved by gel filtration, althoughpre-treatment at 37° C. induces protein aggregation and elution from asize exclusion column in the column void. Interestingly, carefulanalysis of the kinetics of inactivation of both T172R/G173Q and L169Kreveal that the activity diminishes exponentially in the first phase theactivity but appears to reach a plateau at approximately 35% of theirmaximal activity. This activity remains even after greater than 8 hr, incontrast to wt-CocE, G173Q and T172R where the activity decays to lessthan 10% activity within 90 min. One plausible explanation for thisbehavior (of T172R/G173Q and L169K) is that there still remains athermally-sensitive portion of the enzyme that does not result incomplete enzyme inactivation. The remaining thermally-sensitive regionmay be located at the dimer interface, the disruption of which couldlead to aggregation. The gel filtration chromatography data mentionedearlier would concur with this notion. Indeed, further studies and thecharacterization of additional mutants within the dimer interface areongoing.

Perhaps the most dramatic effect of the mutations is revealed by theirin vivo ability to protect against cocaine-induced lethality. The effectof the mutations paralleled our in vitro data through extending theduration of pretreatment that the enzyme is capable of withstanding. Inthe case of T172R/G173Q the pretreatment duration in which the enzymewas still capable of protecting against cocaine-induced lethality by 50%was extended by greater than 20-fold, or up to 4.5 hours. This stronglysuggests that the cause of the in vivo instability of CocE is the sameas that observed for the enzyme in vitro.

In summary, the present work shows that a multi-pronged approachcombining computational, biochemical and structural analysis can be usedto rationally develop variants of CocE that are significantly morestable than the native enzyme.

Example 5 CocE Mutant L169K/G173Q

A mutant of CocE, L169K/G173Q, was created and characterized as follows.

Design of Thermostable Mutations.

Based on the X-ray crystal structure (PDB code 1JU3) of the bacterialcocaine esterase (CocE) (Larsen et al., 2002), a complete 3D model ofCocE bound to (−)-cocaine was built using energy minimizations andmolecular dynamics (MD) simulations encumbered within the AMBER program(Case et al., 2004)(See Example 4 above). To increase thethermostability of CocE, a computational method implemented in theRosettaDesign program was used (Kuhlman and Baker, 2000; Korkegian etal., 2005). This method was capable of predicting thermostabilizingmutations within a given fold while minimizing any shift in the backbonethat might structurally disrupt the active site structure or quench itsflexibility. The method implemented in the RosettaDesign program uses anenergy function for evaluating the fitness of a particular sequence fora given fold and a Monte Carlo search algorithm for sampling sequencespace. The same method has successfully been used by other researchersto increase thermostability of an enzyme with no reduction in catalyticefficiency (Kuhlman and Baker, 2000; Korkegian et al., 2005). Thecomputational modeling using the RosettaDesign program has allowed theprediction of a set of mutations that can potentially lower the energyand, therefore, increase the higher thermostability of CocE. As thefirst round of the rational design, the computation was simplified byonly considering the possible mutations on the amino acid residues thathave a distance of 6-25 Å from the catalytic site.

Site Directed Mutagenesis.

Point mutations at positions 169 and 173 were introduced into the CocEcDNA cloned in the bacterial expression vector, pET-22b(+). Mutationswere generated using a modified QuickChange (Stratagene) mutagenesisprotocol and single oligonucleotide primers. For generation of thedouble mutant, cDNAs with the single G173Q point mutation was used as atemplate for the second round of mutagenesis to introduce L169K. Themutant was sequenced in both directions over the entire coding region.Wild-type and CocE mutants were expressed as C-terminally-6× His-taggedproteins in E. coli BL-21 Gold (DE3) cells grown at 37° C. Proteinexpression was induced with 1 mM isopropyl-β-thiogalactopyranoside(IPTG, Fisher) for 12 hours at 18° C.

Purification of Cocaine Esterase and Mutants.

Cells were pelleted, resuspended in 50 mM Tris pH 8.0, 150 mM NaCl withprotease inhibitors (3 μg/ml each of leupeptin and lima bean or soybeantrypsin inhibitor) and lysed using a French press (Thermo FisherScientific Corp, USA). Wild-type or mutant CocE was enriched using Talonmetal affinity chromatography (Clontech Laboratories, Inc, Mountain ViewCalif.), followed by anion-exchange (Q-Sepharose, GE Healthcare,Piscataway N.J.) chromatography. CocE was eluted from the Q-Sepharosecolumn with 150-450 mM NaCl linear gradient buffers containing 20 mMHepes pH 8.0, 2 mM MgCl₂, 1 mM EDTA and 1 mM DTT. The peak fractionswere pooled and concentrated by Centricon-30 (Millipore), snap frozen inliquid nitrogen and stored at −80° C.

Determination of Catalytic Efficiency.

To determine the catalytic activity a spectrophometric assay wasperformed. Samples of L169K/G173Q CocE were added to a 96-well UVpermeable plate containing increasing cocaine concentrations (0.5, 2.5,5, 12.5, 25, 50, 100, and 150 μM) to give a final concentration of 10ng/ml CocE in a final volume of 200 μl. The change in absorbance at 240nm was measured over 20 minutes, with readings every 10 seconds, by aSpectraMax Plus 384 UV plate reader (Molecular Devices, Sunnyvale,Calif.) using SOFTmax Pro software (Version 3.1.2). The change inabsorbance was converted to the change in concentration and furthermorethe rate of decay per mole enzyme is determined (K_(cat)). K_(cat) andK_(m) of the enzyme are determined using Prism (GraphPad software, SanDiego). The L169K/G173Q mutation allows each molecule of the enzyme toturn over approximately 6000 molecules of cocaine per minute intoinactive metabolites. The increase in K_(cat) over the wildtype and theprevious T172R/G173Q mutation is accompanied by an increase in K_(m),which results in a similar catalytic efficiency to both the wild typeand T172R/G173Q mutation (FIG. 34).

Determination of In Vitro Half Life.

To mimic body temperature and enzyme concentration in the NIH Swissmouse, CocE was incubated in a 37° C. water bath at a concentration of60 μg/ml in either human plasma or phosphate buffered saline (PBS) pH7.4. The samples were added to the 37° C. water directly from −80° C.storage at varying times (0, 24, 48, 77, 96, 120 hours) and all wereassayed at a final concentration of 10 ng/ml as described above.

The substitution of a lysine and a glutamate and positions 169 and 173respectively extends the in vitro half life to approximately 72 hours,which is 332 times longer than the wild type enzyme and 17 times longerthan the T172R/G173Q mutation (FIG. 35).

Determination of In Vivo Potency.

The increase in K_(cat) has been shown in vivo as well. The L169K/G173Qmutated CocE dose-dependently protected mice from increasing lethaldoses of cocaine (FIG. 36). CocE was administered IV into the tail veinof NIH Swiss mice in a volume of 0.2 ml. Varying concentrations ofcocaine were delivered into the intraperitoneal cavity 1 min later. Thismutant has shown to be more potent than previous CocE mutants,maintaining some degree of protection at doses as low as 0.01 mg. Theincreased potency should allow less enzyme to be used and thereforeshould decrease the innate immunological responses of the animals to theprotein. Increased potency also makes this enzyme more effective againstextreme doses of cocaine that may be seen in a human overdose, atequivalent concentrations to other enzyme mutation.

Evaluation of In Vivo Half Life.

CocE is administered IV to the tail vein in a volume of 0.2 ml. Animalsare challenged with 180 mg/kg cocaine delivered intraperitoneally at agiven time after CocE administration. The L169K/G173Q mutant of CocE wastested at 1 mg against the other mutants because 1 mg of CocE showed thegreatest separation between in vivo thermostabilities of different CocEmutants in preliminary studies (data not shown). The L169K/G173Q mutantof CocE (1 mg) protected 50% of NIH Swiss mice from death for up to 7.5hrs. Lower does of the L169K/G173Q mutation show extended protectionagainst lethality as compared to previous mutations as well (FIG. 37).

Rhodococcus CocE amino acid sequence SEQ ID NO: 1   1mvdgnysvas nvmvpmrdgv rlavdlyrpd adgpvpvllv  rnpydkfdvf awstqstnwl  61efvrdgyavv iqdtrglfas egefvphvdd eadaedtlsw  ileqawcdgn vgmfgvsylg 121vtqwqaaysg vgglkaiaps masadlyrap wygpggalsv  eallgwsali gtglitsrsd 181arpedaadfv qlaailndva gaasvtplae qpllgrlipw  vidqvvdhpd ndeswqsisl 241ferlgglatp alitagwydg fvgeslrtfv avkdnadarl  vvgpwshsnl tgrnadrkfg 301iaatypiqea ttmhkaffdr hlrgetdala gvpkvrlfvm  gidewrdetd wplpdtaytp 361fylggsgaan tstgggtlst sisgtesadt ylydpadpvp  slggtllfhn gdngpadqrp 421ihdrddvlcy stevltdpve vtgtvsarlf vsssavdtdf  taklvdvfpd graialcdgi 481vrmryretiv nptlieagei yevaidmlat snvflpghri  mvqvsssnfp kydrnsntgg 541viareqleem ctavnrihrg pehpshivlp iikr

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In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references.

What is claimed is:
 1. A composition comprising: a cocaine esterase(CocE) polypeptide; and at least one thermostabilization compound,wherein the CocE in the presence of the compound is more thermostablethan the CocE in the absence of the compound.
 2. The composition ofclaim 1, wherein the CocE comprises: (i) an amino acid sequence of SEQID NO: 1; (ii) an amino acid sequence at least 90% identical to SEQ IDNO: 1 having esterase activity; or (iii) an amino acid sequence at least90% identical to SEQ ID NO: 1 with one or more substitutions selectedfrom the group consisting of L163V, V225I, I218L, A310D, A149S, S159A,S265A, S56G, W220A, S140A, F189L, A193D, T254R, N42V, V262L, L508G,Y152H, V160A, T172R, Y532F, T74S, W285T, L146P, D533S, A194R, G173Q,C477T, K531A, R41I, L119A, K46A, F84Y, T172R/G173Q, L169K, F189A, N197K,R182K, F189K, V190K, Q191K, A194K, and L169K/G173Q, or a combinationthereof; and having esterase activity.
 3. The composition of claim 2,wherein the CocE has an amino acid sequence of SEQ ID NO: 1 with one ormore substitutions selected from the group consisting of: T172R, S159A,N197K, L169K, F189K, G173Q, and T172R/G173Q.
 4. The composition of claim2, wherein the CocE has an amino acid sequence of SEQ ID NO:1 with thesubstitution L169K/G173Q.
 5. The composition claim 1, wherein the CocEis a pegylated CocE.
 6. The composition of claim 1, wherein the one ormore thermostabilization compounds are selected from:


7. The composition of claim 1, wherein the at least onethermostabilization compound is selected from:


8. The composition of claim 1, wherein the at least onethermostabilization compound is:


9. The composition of claim 1, wherein the at least onethermostabilization compound is:


10. The composition of claim 1 further comprising a pharmaceuticallyacceptable carrier.
 11. The composition of claim 1, wherein the CocEcomprises (i) an amino acid sequence of SEQ ID NO: 1; (ii) an amino acidsequence at least 90% identical to SEQ ID NO: 1 having esteraseactivity; or (iii) an amino acid sequence at least 90% identical to SEQID NO: 1 with one or more substitutions selected from the groupconsisting of L163V, V225I, I218L, A310D, A149S, S159A, S265A, S56G,W220A, S140A, F189L, A193D, T254R, N42V, V262L, L508G, Y152H, V160A,T172R, Y532F, T74S, W285T, L146P, D533S, A194R, G173Q, C477T, K531A,R41I, L119A, K46A, F84Y, T172R/G173Q, L169K, F189A, N197K, R182K, F189K,V190K, Q191K, A194K, and L169K/G173Q, or a combination thereof; andhaving esterase activity; the one or more thermostabilization compoundsare selected from:

and the composition further comprises a pharmaceutically acceptablecarrier.
 12. A method of thermostabilizing a cocaine esterase (CocE),the method comprising combining a CocE polypeptide with one or morethermostabilization compounds selected from:


13. The method of claim 12, wherein the CocE polypeptide comprises: (i)an amino acid sequence of SEQ ID NO: 1; (ii) an amino acid sequence atleast 90% identical to SEQ ID NO: 1 having esterase activity; or (iii)an amino acid sequence at least 90% identical to SEQ ID NO: 1 with oneor more substitutions selected from the group consisting of L163V,V225I, I218L, A310D, A149S, S159A, S265A, S56G, W220A, S140A, F189L,A193D, T254R, N42V, V262L, L508G, Y152H, V160A, T172R, Y532F, T74S,W285T, L146P, D533S, A194R, G173Q, C477T, K531A, R41I, L119A, K46A,F84Y, T172R/G173Q, L169K, F189A, N197K, R182K, F189K, V190K, Q191K,A194K, and L169K/G173Q, or a combination thereof; and having esteraseactivity.
 14. The method of claim 12, wherein combining the CocEpolypeptide with one or more thermostabilization compounds occurs (i) invivo in a mammal, (ii) in vitro, (iii) during purification of CocE, (iv)during storage of CocE, or a combination thereof.
 15. A method oftreating a cocaine-induced condition, the method comprising:administering to a mammalian subject in need thereof a compositionselected from: (i) a composition according to claim 1; or (ii)composition comprising an isolated cocaine esterase (CocE) polypeptidehaving (a) an amino acid sequence of SEQ ID NO:1, except forsubstitutions L169K and G173Q; or (b) an amino acid sequence having atleast 85% sequence identity with SEQ ID NO:1, having substitutions L169Kand G173Q; esterase activity; and increased thermostability at 37° C. ascompared to wild-type CocE; wherein the subject is diagnosed with, or atrisk for, a cocaine-induced condition selected from the group consistingof cocaine overdose, cocaine toxicity, cocaine addiction, or cocainedependence.
 16. The method of claim 15, wherein the CocE polypeptidecomprises: (i) an amino acid sequence of SEQ ID NO: 1; (ii) an aminoacid sequence at least 90% identical to SEQ ID NO: 1 having esteraseactivity; or (iii) an amino acid sequence at least 90% identical to SEQID NO: 1 with one or more substitutions selected from the groupconsisting of L163V, V225I, I218L, A310D, A149S, S159A, S265A, S56G,W220A, S140A, F189L, A193D, T254R, N42V, V262L, L508G, Y152H, V160A,T172R, Y532F, T74S, W285T, L146P, D533S, A194R, G173Q, C477T, K531A,R41I, L119A, K46A, F84Y, T172R/G173Q, L169K, F189A, N197K, R182K, F189K,V190K, Q191K, A194K, and L169K/G173Q, or a combination thereof; andhaving esterase activity.
 17. The method of claim 15, wherein the atleast one thermostabilization compound is selected from:


18. The method of claim 15, wherein the composition is administeredintravenously or via a mini-infusion pump.
 19. A method for identifyinga thermostabilization compound, the method comprising: measuringthermostability of a cocaine esterase (CocE); measuring thermostabilityof the CocE in combination with a candidate compound; determiningwhether the candidate compound increases thermostability of the CocE.20. A composition comprising: (i) an isolated cocaine esterase (CocE)polypeptide, the CocE polypeptide comprising: (a) an amino acid sequenceof SEQ ID NO:1, except for substitutions L169K and G173Q; or (b) anamino acid sequence having at least 85% sequence identity with SEQ IDNO:1, wherein the encoded CocE polypeptide has substitutions L169K andG173Q and esterase activity with increased thermostability at 37° C. ascompared to wild-type CocE; or (ii) an isolated nucleic acid encodingthe polypeptide of (i); and (iii) optionally, a pharmaceuticallyacceptable carrier.